- Recognize the effects of Oxygen on bacteria
- Explain the various oxygen requirements of the microbes, observe and interpret the growth of microbes in thioglycollate agar deep media
- Discuss methods of culturing anaerobic bacteria
Environmental Requirements: Oxygen requirements
How does Oxygen affect bacterial growth?
Bacteria can differ dramatically in their ability to utilize oxygen (O2). Under aerobic conditions, oxygen acts as the final electron acceptor for the electron transport chain located in the plasma membrane of prokaryotes. Bacteria use this process to generate ATP, the energy source for most cellular processes. In the absence of oxygen (O2), some bacteria can use alternative metabolic pathways including anaerobic respiration and/or fermentation. During anaerobic respiration, other alternative molecules are used as the final electron acceptor for the electron transport chain such as nitrate (NO3), sulfate (SO4), and carbonate (CO3).
Bacteria and many microorganisms are very sensitive to oxygen concentrations. Some will only grow in its presence and are called obligate aerobes. Facultative aerobes will grow either aerobically or in the absence of oxygen (anaerobic conditions), but they generally do better with oxygen. Aerotolerant anaerobes don't require oxygen, but can grow in its presence, while strict obligate anaerobes cannot use oxygen and cannot grow or survive in its presence. Microaerophiles use oxygen, but at lower concentrations than atmospheric oxygen levels (which is ~20%).
One can determine a bacterium's oxygen requirements by cultivating them in a special medium called thioglycollate agar tubes. The bottom of the tube of medium is kept anaerobic by cystine and thioglycollic acid, which chemically react with, and tie up any oxygen that diffuses in. Any un-reacted oxygen in the tube will be indicated by resazurin, a dye that turns pink in the presence of oxygen. It is common for the top centimeter or so to be pink. One can inoculate a thioglycollate tube with your bacterium and observe where the bacterium grows in the tube to determine its oxygen requirements (see Image 1)
Image 1: Microbial oxygen requirements determined using thioglycollate agar tubes. Green dots represent bacterial colonies within in the agar or on its surface. The surface of the agar tube is directly exposed to atmospheric oxygen, and will be aerobic. The oxygen content of the thioglycollate medium decreases with depth until the medium becomes anaerobic towards the bottom of the tube.
Cultivation of Anaerobes
The cultivation of anaerobes can be done in anaerobic chamber (image 2). This is a special chamber where you can work with and cultivate strict obligate anaerobes without exposing them to oxygen. Anaerobic chambers contain a hydrogen (H2) gas mixture that is circulated through a heated palladium catalyst to remove oxygen (O2) by forming water (H2O). Anaerobic chambers use a gas mixture of H2 and nitrogen gas (N2) (5/95%) or N2/carbon dioxide (CO2)/H2 (85/10/5 %) to remove oxygen. An airlock is used to reduce O2 levels prior to the transfer of samples in and out of the chamber.
Another way of culturing bacteria anaerobically on plates is to use a GasPak anaerobic system. In these systems, hydrogen and carbon dioxide are generated by a GasPak envelope after the addition of water. A palladium catalyst in the chamber of the GasPak system catalyzes the formation of water from hydrogen and oxygen, thereby removing oxygen from the sealed chamber (image 3 and 4). These systems are compact, easy to use, and less expensive than an anaerobic chamber. They come in jars (image 3) or in a box format (image 4).
Image 2:Anaerobic chamber. https://coylab.com/products/anaerobi...robic-chamber/
Image 3: GasPak system jars Image 4: GasPak system boxes. https://www.fishersci.com/shop/produ...rs-3/p-4902079
Watch Video 1: Basics of thioglycollate media
Watch Video 1: explanation on how thioglycollate media works and examples. (9:33) URL: https://youtu.be/AJG18sQd8mU
Watch Video 2: how to prepare an anaerobic jar
Watch Video 2: how to set up an anaerobic jar. The process is similar for an anaerobic box. (3:03) URL: https://youtu.be/aFDYx-7ceS8